Gene Cloning and Nucleotide Sequence of Human Dihydrolipoamide Dehydrogenase-Binding Protein

  • Lee, Jeongmin (Department of Genetic Engineering, College of Life Science and Technology, Sungkyunkwan University) ;
  • Ryou, Chongsuk (Institute for Neurodegenerative Diseases, University of California San Francisco) ;
  • Jeon, Bong Kyun (Department of Genetic Engineering, College of Life Science and Technology, Sungkyunkwan University) ;
  • Lee, Poongyeon (Department of Genetic Engineering, College of Life Science and Technology, Sungkyunkwan University) ;
  • Woo, Hee-Jong (Department of Immunology, School of Veterinary Medicine, Seoul National University) ;
  • Kwon, Moosik (Department of Genetic Engineering, College of Life Science and Technology, Sungkyunkwan University)
  • Received : 2001.04.27
  • Accepted : 2001.09.21
  • Published : 2002.03.01


The pyruvate dehydrogenase complex (PDC), a member of $\alpha$-keto acid dehydrogenase complex, catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and $H^+$. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, we have partially cloned the gene for E3BP in human. Nine putative clones were isolated by human genomic library screening with 1.35 kb fragment of E3BP cDNA as a probe. For investigation of cloned genes, Southern blot analysis and the construction of the restriction map were performed. One of the isolated clones, E3BP741, has a 3 kb-SacI fragment, which contains 200 bp region matched with E3BP cDNA sequences. The matched DNA sequence encodes the carboxyl-terminal portion of lipoyl-bearing domain and hinge region of human E3BP. Differences between yeast E3BP and mammalian E3BP coupled with the remarkable similarity between mammalian E2 and mammalian E3BP were confirmed from the comparison of the nucleotide sequence and the deduced amino acid sequence in the cloned E3BP. Cloning of human E3BP gene and analysis of the gene structure will facilitate the understanding of the role(s) of E3BP in mammalian PDC.


  1. Harris, R. A., M. Bowker-Kinley, P. Wu, J. Jeng and K. M. Popov. 1997. Dihydrolipoamide dejydrogenase-binding protein of the human pyruvate dehydrogenase complex. J. Biol. Chem. 272:19746-19751.
  2. Lee, S. H. and J. B. Clark. 1997. High-yield method for isolation of $\lambda$ DNA. Biotechniques 23:589-589.
  3. Maeng, C. Y., M. A. Yazdi and L. J. Reed. 1996. Stoichiometry of binding of mature and truncated forms of the dihydrolipoamide dehydrogenase-binding protein to the dihydrolipoamide acetyltransferase core of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae. Biochemistry 35:5879-5882.
  4. Hodgson, J. A., O. G. De Marcucci and J. G. Lindsay. 1986. Lipoic acid is the site of substrate-dependent acetylation of component X in ox heart pyruvate dehydrogenase multienzyme complex. Eur. J. Biochem. 158:595-600.
  5. Tan, J. and M. S. Patel. 1999. Cloning and characterization of a 5.9 kb promoter region of the human pyruvate dehydrogenase $\alpha$ subunit gene. Biochem. Biophys. Acta 1431:531-537.
  6. Lee, J., C. Ryou and M. Kwon. 2001. Molecular cloning and expression of human dihydrolipoamide dehydrogenase-binding protein in Escherichia coli. J. Microbiol. Biotechnol. 11:592-597.
  7. Neagle, J., O. De Marcucci, B. Dunbar and J. G. Lindsay. 1989. Component X of mammalian pyruvate dehydrogenase complex: structural and functional relationship to the lipoate acetyltransferase (E2) component. FEBS Letter 253:11-15.
  8. Roche, T. E. and M. S. Patel. 1989. Alpha-keto acid dehydrogenase complexes: organization, regulation, and biomedical ramification. Ann. N.Y. Acad. Sci. 573:1-474.
  9. Klingbeil, M. M., D. J. Walker, R. Arnette, E. Sidawy, K. Hayton, P. R. Komuniecki and R. Komuniecki. 1996. Identification of a novel dihydrolipoyl dehydrogenase-binding protein in the pyruvate dehydrogenase complex of the anaerobic parasitic nematode, Ascaris suum. J. Biol. Chem. 271:5451-5457.
  10. Patel, M. S. and T. E. Roche. 1990. Molecular biology and biochemistry of pyruvate dehydrogenase complexes. FASEB J. 4:3224-3233.
  11. Patel, M. S. and R. A. Harris. 1995. Mammalian alpha-keto acid dehydrogenase complexes: gene regulation and genetic defects. FASEB J. 9:1164-1172.
  12. Lim, J. Y., C. S. Shin, E. J. Chung, J. S. Kim, H. U. Kim, S. J. Oh, W. B. Choi, C. S. Ryou, J. B. Kim, M. S. Kwon, T. Y. Chung, S. I. Song, S. I. Kim, B. H. Nahm, Y. S. Hwang, M. Y. Eun, J. S. Lee, J. J. Cheong and Y. D. Choi. 2000. Analysis of expressed sequence tags from Brassica rapa L. ssp. pekinensis. Mol Cells. 10:399-404.
  13. Hodgson, J. A., O. G. De Marcucci and J. G. Lindsay. 1988. Structure function studies on the lipoate-acetyltransferasecomponent- X-core assembly of the heart pyruvate dehydrogenase complex. Eur. J. Biochem. 171:609-614.
  14. Sanderson, S. J., C. Miller and J. G. Lindsay. 1996. Stoichiometry, organisation and catalytic function of protein X of the pyruvate dehydrogenase complex from bovine heart. Eur. J. Biochem. 236:68-77.
  15. Roche, T. E., M. Rahmatullah, S. L. Powers-Greenwood, G. A. Radke, S. Gopalakrishnan and C. L. Chang. 1989. The lipoylcontaining components of the mammalian pyruvate dehydrogenase complex: structural comparison and subdomain roles. Ann. N. Y. Acad. Sci. 573:66-75.
  16. Sambrook, J., E. F. Fritsch and T. Maniatis. 1989. Molecular cloning; A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
  17. Sanderson, S. J., S. S. Khan, R. G. McCartney, C. Miller and J. G. Lindsay. 1996. Reconstitution of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes: analysis of protein X involvement and interaction of homologous and heterologous dihydrolipoamide dehydrogenases. Biochem. J. 319:109-116.